PROJECT SUMMARY Zika virus (ZIKV) (Flaviviridae, flavivirus) is a serious emerging threat in the US and world-wide, creating an urgent need for accurate, Point-of-Care (POC) diagnostic tests. The project goal is to develop two novel, complementary FDA-cleared (510k) Lateral Flow Immunoassay (LFI) devices: 1) a zNS1-LFI to diagnose infection early by detecting ZIKV-specific epitopes on ZIKV NS1 (zNS1), a protein released at high levels during viremia and 2) a zNS1-serology LFI to diagnose past infection, by detecting antibodies to ZIKV- specific epitopes on zNS1. The tests will be rapid, affordable, easy to use at POC and provide patients in developed and developing nations access to actionable diagnostic information. Phase I will create novel monoclonal antibodies (mAbs) that recognize ZIKV-specific epitopes on zNS1. These mAbs will be used to make a prototype zNS1-LFI that detects zNS1 with high sensitivity and does not cross-react with NS1 proteins of other common flaviviruses. Clinical utility proof of concept will be demonstrated using a new nonhuman primate (NHP) model of ZIKV infection to show that the zNS1-LFI can diagnose acute ZIKV infection using easily obtained samples such as blood, saliva or urine. The NHP model will then be used to test the hypothesis that fetal ZIKV infection is associated with atypical, persistent presence of zNS1 in the blood of infected pregnant females. Positive results would be a significant first step to provide pregnant women in ZIKV-affected regions with a rapid, inexpensive method to diagnose the risk of fetal ZIKV infection. Phase II will optimize the prototype zNS1-LFI and generate more detailed clinical utility data using the CNPRC NHP model. Phase II work will also generate analytical and clinical (human) performance data to support applications for an FDA Emergency Use Authorization (EUA) to allow immediate use and concurrently an application will be submitted to FDA for 510(k) clearance as a POC IVD. zNS1 epitopes validated as ZIKV- specific in Phase I will be used in Phase II to make a novel ZIKV-specific protein construct as the key detector component in a ZIKV-specific zNS1-serology LFI. Analytical and clinical (human) performance data will then be collected to support applications for FDA EUA and FDA 510(k) clearance as a POC IVD to detect past ZIKV infection. Development of these ZIKV-specific tests will be a significant advance over existing technologies. LFI is a proven, highly reliable, robust and extremely simple diagnostic assay platform, well-suited for use in resource-limited areas bearing the brunt of the current Zika outbreak. In contrast, all ZIKV diagnostic tests approved to date under FDA EUA are complex and in accordance with EUA, must be performed by highly trained personnel using specialized equipment in centralized clinical reference labs. Therefore, the novel ZIKV-specific POC diagnostic tests proposed here will transform current ZIKV diagnostic practices by providing millions of people with immediate access to affordable diagnostic test results, even in resource-limited settings.